All organisms modify their RNA after transcription, yet both the physiological importance of particular modified nucleosides and the mechanisms by which they are generated remain unknown. The mechanistic elucidation of the generation of two such modified nucleosides will be undertaken: pseudouridine (the most common) and 4-thiouridine (which protects bacteria against exposure to UV light). Pseudouridine synthases convert uridine residues in RNA into pseudouridine, the C-glycoside isomer. There are many such enzymes in all organisms, and they fall into six families. Mutation or deletion of one human pseudouridine synthase, dyskerin, leads to the disease dyskeratosis congenita, and the widespread occurrence and conservation of pseudouridine residues in RNA attests to their physiological importance. The new investigations extend our success towards the elucidation of the chemical mechanism followed by the pseudouridine synthases and ThiI, the enzyme responsible for 4-thiouridine generation. Knowledge of the mechanisms is valuable in its own right as a chemical precedent and because it may allow the more efficient development of pharmaceuticals that specifically targets these enzymes in pathogenic bacteria. In particular, recent results strongly disfavor one of the two mechanisms that had dominated thought on how pseudouridine is formed and simultaneously suggest a previously unrecognized possibility. This new alternative will be tested using kinetic isotope effect studies. The preference of the pseudouridine synthase RluA for its various substrates will also be investigated to probe the mode of molecular recognition that allows this enzyme to distinguish substrate from non-substrate RNA. A novel mechanism for 4-thiouridine generation involving a disulfide-linked adduct between ThiI and RNA will be tested by fully characterizing a species that match its properties in preliminary experiments. The identity of new mechanistically important amino acid residues of ThiI will also be sought. These projects will be undertaken by graduate students and by undergraduate researchers to expose them to cutting-edge research methods and thought.